ACTIONS OF SEMINAL PROTEINS IN MATED DROSOPHILA FEMALES

精液蛋白在交配雌性果蝇中的作用

• 批准号: 6651927
• 负责人: Mariana Federica Wolfner
• 金额: $ 4.38万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6651927
• 关键词: Drosophilidae; arthropod genetics; biological signal transduction; cytotoxicity; female; female reproductive system; fertilization; gene expression; gene mutation; genetic screening; glycoproteins; green fluorescent proteins; immunoprecipitation; ovulation; peptide hormone biosynthesis; protease inhibitor; protein structure function; proteolysis; semen; sperm; tissue /cell culture; trypsin inhibitors

Far from being a passive medium for sperm delivery, seminal fluid contains proteins that exert major influences on mated females. This is best understood in insects, where seminal fluid proteins ("Acps") stimulate ovulation and egg-laying, are required for a female to store sperm, and decrease her lifespan. Despite their importance, the molecular actions of seminal fluid proteins are unknown. We will use the tractable molecular genetics of Drosophila to determine how three major reproductive processes are controlled by Acps. In aim 1, we address how ovulation is stimulated by Acp26Aa, a male-derived prohormone. Using an Acp26Aa null mutation, we'll identify signals that transduce the effect of Acp26Aa. We will also identify the receptor through which Acp26Aa acts, and whether Acp26Aa needs processing, and its region similar to mollusk egg-laying hormones, to act. Our results are significant to reproduction, hormone action, and the evolution of reproductive isolation mechanisms. In aim 2, we investigate how seminal fluid proteins cause sperm storage. Again we'll work out from a single molecule, Acp36DE, that we showed genetically is central to this process. We will examine the behavior of GFP-labeled sperm in the absence of Acp36DE, testing whether Acp36DE acts within the sperm storage organs or to get sperm into storage. We will test whether Acp36DE's function requires its binding to sperm, and will identify molecules to which Acp36DE binds. Sperm storage is widespread among animals; our study will determine its molecular basis. In aim 3, we address the role of protease inhibitors in seminal fluid. Seminal fluid of many animals contains protease inhibitors, but their reproductive function is unknown. Acp62F is a trypsin inhibitor in Drosophila seminal fluid. Intriguingly, Acp62F is toxic when introduced into insect circulatory systems. We propose a mutational analysis to determine Acp62F's function in the seminal fluid, such as controlling proteolysis of crucial reproductive proteins. We will test if entry of Acp62F into the female's circulatory system during mating shortens her life. We will identify molecules which bind Acp62F; these should include the proteases it inhibits. Our results will establish reproductive roles for controlled proteolysis, and will test evolutionary hypotheses for why toxic proteins exist in semen. In aim 4, we'll continue screening a collection of EMS-induced mutations for ones in Acps. Analyzing these mutants, and ectopic expression of Acps, will identify new Acps that regulate ovulation, sperm storage or proteolysis.

精液不仅不是传递精子的被动媒介，还含有对交配的雌性产生重大影响的蛋白质。这一点在昆虫身上理解得最清楚，在昆虫中，精液蛋白(“ACP”)刺激排卵和产卵，是雌性储存精子所必需的，并降低了她的寿命。尽管它们很重要，但精液蛋白的分子作用尚不清楚。我们将利用果蝇易处理的分子遗传学来确定ACPs如何控制三个主要的生殖过程。在目标1中，我们讨论了Acp26Aa是如何刺激排卵的，Acp26Aa是一种男性衍生的前激素。使用Acp26Aa零突变，我们将识别转导Acp26Aa效应的信号。我们还将确定Acp26Aa通过哪个受体起作用，Acp26Aa是否需要处理，以及它的类似于软体动物产卵激素的区域才能起作用。我们的结果对生殖、激素作用和生殖隔离机制的进化具有重要意义。在目标2中，我们研究精液蛋白是如何导致精子储存的。再一次，我们将从单个分子Acp36DE算出，我们证明了基因在这个过程中是核心的。我们将检查在没有Acp36DE的情况下GFP标记的精子的行为，测试Acp36DE是在精子存储器官内发挥作用，还是将精子送入存储。我们将测试Acp36DE的功能是否需要它与精子结合，并将确定Acp36DE结合的分子。精子储存在动物中很普遍；我们的研究将确定其分子基础。在目标3中，我们讨论了精液中蛋白水解酶抑制剂的作用。许多动物的精液中含有蛋白酶抑制剂，但它们的生殖功能尚不清楚。Acp62F是果蝇精液中的一种胰酶抑制剂。有趣的是，当Acp62F进入昆虫循环系统时，它是有毒的。我们建议进行突变分析，以确定Acp62F在精液中的功能，如控制关键生殖蛋白的蛋白质分解。我们将测试交配期间Acp62F进入雌性的循环系统是否会缩短她的寿命。我们将确定结合Acp62F的分子；这些分子应该包括它抑制的蛋白水解酶。我们的结果将确立受控蛋白分解的生殖作用，并将检验为什么有毒蛋白存在于精液中的进化假说。在目标4中，我们将继续筛选EMS诱导的ACPs突变集合。分析这些突变体和ACP的异位表达，将发现新的ACP调控排卵、精子储存或蛋白质分解。